Project Summary/Abstract Lung cancer progression involves coordinate changes in both oncogene and tumor suppressor function. Protein Phosphatase 2A (PP2A) is a tumor suppressor that is dysregulated and deactivated in lung cancer. PP2A is inactivated through several mechanisms including somatic mutations, suppression of individual subunits, increased expression of endogenous PP2A inhibitors and changes in post-translational modifications of the C subunit. While several therapeutics have been developed to indirectly target PP2A, recent studies have uncovered small molecules such as phenothiazines that directly activate PP2A. Nevertheless, because their pronounced extrapyramidal and anti-cholinergic effects were severely dose limiting, further pursuit of phenothiazines and related dibenzazepines for treatment of cancer seems unlikely. Our lab has reengineered the tricyclic neuroleptics to decouple the CNS pharmacology from the anti-proliferative properties of this drug- class. We have developed and characterized these small molecule activators of PP2A (SMAPs) as both chemical tools to probe PP2A regulation and for the treatment of PP2A dependent diseases. Our combined structure- function studies have revealed that SMAPs bind to PP2A in a way that protects the regulatory C-terminal tail of the catalytic subunit and stabilizes the holoenzyme. Our goal is to define the SMAP induced alterations on PP2A holoenzyme composition that promote the tumor suppressive properties of PP2A. We aim to identify the tumor suppressive PP2A regulatory subunits enhanced by SMAPs in order to correlate treatment response to clinical and genetic characteristics, and to aid in the design of more selective, next-generation SMAPs. In this project, three robust aims are designed to assess how activation of PP2A using SMAPs represents an effective treatment option for patients diagnosed with NSCLC. Aim 1 will structurally and functionally characterize the physiologic and cancer-relevant posttranslational regulation of PP2A by SMAPs. We will investigate the SMAPs-specific structural modulation of the PP2A holoenzyme by various, recurrent tumor derived mutations through coupling methyl-PP2A-C antibody and immunoprecipitation-mass spectrometry based approaches to quantify post- translational modification and PP2A activity. Aim 2 will discern how PP2A A? mutations modulate the biological activity of SMAPs. We will assess the functional role of common missense mutations in lung cancer by using isogenic CRISPR/Cas9 cell lines to identify cellular phenotypes, subunit expression, SMAPs response, and biological responses in nude mice. Aim 3 will determine combination treatment strategies to promote regression of tumors in KRAS mutant lung cancer. We will examine rational treatment combinations with SMAPs based on our studies demonstrating that protein phosphatase 2A (PP2A) activity defines the response of KRAS mutant lung cancer cells across library of >200 kinase inhibitors. Collectively, these studies will reveal novel insights into the molecular basis of regulation of PP2A and functional consequences of PP2A activation/inactivation in lung cancer.